Xia, XG, Zhou, H, Samper, E, Melov, S and Xu, Z. Pol II-expressed shRNA knocks down Sod2 gene expression and causes phenotypes of the gene knockout in mice. PLoS Genet 2: e10

Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.
PLoS Genetics (Impact Factor: 7.53). 02/2006; 2(1):e10. DOI: 10.1371/journal.pgen.0020010
Source: PubMed


RNA interference (RNAi) has been used increasingly for reverse genetics in invertebrates and mammalian cells, and has the potential to become an alternative to gene knockout technology in mammals. Thus far, only RNA polymerase III (Pol III)-expressed short hairpin RNA (shRNA) has been used to make shRNA-expressing transgenic mice. However, widespread knockdown and induction of phenotypes of gene knockout in postnatal mice have not been demonstrated. Previous studies have shown that Pol II synthesizes micro RNAs (miRNAs)-the endogenous shRNAs that carry out gene silencing function. To achieve efficient gene knockdown in mammals and to generate phenotypes of gene knockout, we designed a construct in which a Pol II (ubiquitin C) promoter drove the expression of an shRNA with a structure that mimics human miRNA miR-30a. Two transgenic lines showed widespread and sustained shRNA expression, and efficient knockdown of the target gene Sod2. These mice were viable but with phenotypes of SOD2 deficiency. Bigenic heterozygous mice generated by crossing these two lines showed nearly undetectable target gene expression and phenotypes consistent with the target gene knockout, including slow growth, fatty liver, dilated cardiomyopathy, and premature death. This approach opens the door of RNAi to a wide array of well-established Pol II transgenic strategies and offers a technically simpler, cheaper, and quicker alternative to gene knockout by homologous recombination for reverse genetics in mice and other mammalian species.

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    • "For experiments involving siRNA synthesis, both RNApol II-and RNApol III-dependent promoters have been used. Constructs containing promoters transcribed by RNApol II are used when a tight regulation of transgene expression is desired (i.e., tissue-specific or inducible expression ) [7] [15]. In general, as RNApol III promoters are shorter than RNApol II promoters, they are more suitable for constructs with length limitations, like those carried by viral vectors. "
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    ABSTRACT: Inhibition of gene expression through siRNAs is a tool increasingly used for the study of gene function in model systems, including transgenic mice. To achieve perdurable effects, the stable expression of siRNAs by an integrated transgenic construct is necessary. For transgenic siRNA expression, promoters transcribed by either RNApol II or III (such as U6 or H1 promoters) can be used. Relatively large amounts of small RNAs synthesis are achieved when using RNApol III promoters, which can be advantageous in knockdown experiments. To study the feasibility of H1 promoter-driven RNAi-expressing constructs for protein knockdown in transgenic mice, we chose IKK1 as the target gene. Our results indicate that constructs containing the H1 promoter are sensitive to the presence of prokaryotic sequences and to transgene position effects, similar to RNApol II promoters-driven constructs. We observed variable expression levels of transgenic siRNA among different tissues and animals and a reduction of up to 80% in IKK1 expression. Furthermore, IKK1 knockdown led to hair follicle alterations. In summary, we show that constructs directed by the H1 promoter can be used for knockdown of genes of interest in different organs and for the generation of animal models complementary to knockout and overexpression models.
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    • "The following three regions in rat VPS35 ORF were selected for constructing shRNA vectors: 5'-GGCTGTGAAAGTTCAATCATTCC-3' (shRNA-886), 5'-CAAGCTGATGGACGCTCTGAAACA-3' (shRNA-887), and 5'-GGACTTCTATGTTATCACCAAAGA-3' (shRNA-888). Individual shRNA was built to target one of the three selected regions using an established protocol 24. Each shRNA was inserted between BamH1 and Nhe1 sites of pacAd5miR-GFP vector (Cell Biolabs, San Diego, CA, USA). "
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    ABSTRACT: Parkinson's disease primarily results from progressive degeneration of dopaminergic neurons in the substantia nigra. Both neuronal toxicants and genetic factors are suggested to be involved in the disease pathogenesis. The mitochondrial toxicant 1-methyl-4-phenylpyridinium (MPP(+)) shows a highly selective toxicity to dopaminergic neurons. Recent studies indicate that mutation in the vacuolar protein sorting 35 (vps35) gene segregates with Parkinson's disease in some families, but how mutation in the vps35 gene causes dopaminergic cell death is not known. Here, we report that enhanced VPS35 expression protected dopaminergic cells against MPP(+) toxicity and that this neuroprotection was compromised by pathogenic mutation in the gene. A loss of neuroprotective functions contributes to the pathogenesis of VPS35 mutation in Parkinson's disease.
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    • "Notably, Grimm et al. [46] further demonstrated that the Pol-III-directed RNAi systems often generate high concentrated siRNA/shRNA which can oversaturate the cellular miRNA pathway, resulting in global miRNA inhibition and cell death. In view of these problems, a Pol-II-mediated intronic miRNA expression system has the advantage of its autoregulation by the cellular RNA splicing, and NMD mechanisms [9, 33, 47], both of which degrade excessive RNA accumulation to prevent possible cytotoxicity. "
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